This proposal represents a continuing effort by our laboratory to understand the mechanism of protein-mediated sugar transport across cell membranes. The broad goal of this proposal is to understand how sugar transport protein oligomerization occurs and how transport protein oligomerization affects transport function. These studies will assist in our long term goals of understanding the physical basis and the cellular regulation of sugar transport and could ultimately be of value in the management of disordered states such as diabetes. Our studies show that the human erythrocyte sugar transport system exists in two catalytically active forms: as a tetramer (major species) and as a dimer (minor species) of GLUT1 proteins. The oligomeric state of the sugar transport protein determines the catalytic properties of transport. Specific Aim 1 asks whether purified tetrameric GLUT1, like erythrocyte-resident GLUT1, is a multisite, allosteric transporter. Our studies suggest that the arrangement of substrate binding sites in tetrameric and dimeric GLUT1 is different. Tetrameric GLUT1 appears to behave as 2 "two-site carriers" that simultaneously expose both sugar influx and sugar efflux sites to substrate. Dimeric GLUT1 behaves as 2 independent "simple carriers" that alternately expose sugar influx and sugar efflux sites to substrate. We test this hypothesis by asking whether purified, tetrameric or dimeric GLUT1 can bind substrates at sugar influx and at sugar efflux sites simultaneously. If our hypothesis is correct, only the tetramer should expose both sites simultaneously. Specific Aim 2 asks if the cryptic cysteines in tetrameric GLUT1 are present as disulfides. Tetrameric GLUT1 contains only 2 free cysteines per GLUT1 monomer, is converted to dimer by reductant but is not two disulfide-linked dimers. Dimeric GLUT1 contains 6 free cysteines per GLUT1 monomer. We use differential carboxymethylation procedures and peptide mapping to identify the cryptic cysteines of the tetramer and to determine whether the cryptic cysteines are internal disulfides, mixed disulfides or are fatty acylated. Specific Aim 3 asks what role the cryptic cysteines of tetrameric GLUT1 play in tetramerization of GLUT1. If modification of a specific cysteine causes tetramerization of GLUT I, deletion of the cysteine should prevent tetramer formation. Each of the 6 GLUT1 cysteines will be mutated. The effects of these mutations on GLUT1 oligomeric structure, ligand binding and sugar transport will be evaluated. If successful, these studies will greatly assist in our long term goal of understanding sequence-specific determinants of sugar transporter higher order structure and function.